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At least 145 records · Page 8Linked to original sources

PubMed2003

Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery.

A topoisomerase-based method was used to clone PCR products encoding 991 of the 1041 open reading frames identified in the genome sequence of the bacterium that causes syphilis, Treponema pallidum subsp. pallidum. Cloning the open reading frames into the univector plasmid system permitted the rapid conversion of the original clone set to other functional vectors containing a variety of promoters or tag sequences. A computational prediction of signal sequences identified 248 T. pallidum proteins that are potentially secreted from the cell. These clones were systematically converted into vectors designed to express the encoded proteins as glutathione-S-transferase fusion proteins. To test the potential of the clone set for novel antigen discovery, 85 of these fusion proteins were expressed from Escherichia coli, partially purified, and tested for antigenicity by using sera from rabbits infected with T. pallidum. Twelve of the 85 proteins bound significant levels of antibody. Of these 12 proteins, seven had previously been identified as T. pallidum antigens, and the remaining five represent novel antigens. These results demonstrate the potential of the T. pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete.

Antigens, Bacterial↗

PubMed1995

A novel Epstein-Barr virus glycoprotein gp150 expressed from the BDLF3 open reading frame.

Analysis of cDNAs mapping to the BamHI D fragment of the Epstein-Barr virus (EBV) genome indicates that the BDLF3 open reading frame, which is predicted to encode a type 1 membrane protein of 234 amino acids, is expressed as an unspliced message. Expression of the open reading frame as a recombinant protein in vaccinia virus reveals a glycoprotein that has both N- and O-linked sugars. Antibodies made to the recombinant protein immunoprecipitate a late glycoprotein with a mobility of approximately 150,000 Da from EBV-producing cells. The glycoprotein is associated with the virion. Antibodies to it appear to react primarily with carbohydrate and do not demonstrate neutralizing activity.

Amino Acid Sequence↗

PubMed1996

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.

Animals↗

PubMed1992

Mls-1 is encoded by the long terminal repeat open reading frame of the mouse mammary tumor provirus Mtv-7.

The murine Mls-1 antigen is the prototype of endogenous superantigens, molecules whose activities lead to deletion of T cells expressing certain T-cell receptor V beta genes from the mature repertoire. However, Mls-1 also stimulates T cells expressing these particular V beta genes (V beta 6, V beta 7, V beta 8.1, and V beta 9) in vitro, making it one of the strongest known T-cell activators. We have recently reported that the Mls-1 gene is closely linked to the endogenous mammary tumor virus Mtv-7. We now demonstrate that Mls-1 is encoded by the open reading frame in the U3 region of the long terminal repeat of Mtv-7. However, control of expression of this molecule seems complex, depending on the promoter used for the transfection experiments. The sequence of the Mtv-7 open reading frame differs from all other known mammary tumor virus open reading frame sequences in the 3' end, suggesting that the T-cell receptor V beta specificity is conferred by the C terminus of the molecule. The predicted structure of the protein encoded by the open reading frame is consistent with a type II transmembrane molecule where the C terminus is extracellular.

Amino Acid Sequence↗

PubMed2026

Auditing bacterial dark-gene screens for superimposed open reading frame artefacts: A multi-layer analysis of Rv2438A in Mycobacterium tuberculosis.

Essentiality and knockdown-vulnerability screens can promote spurious bacterial open reading frames when those frames overlap essential genes, because such a frame inherits its neighbour's signals undiluted and therefore satisfies the screen's criteria better than a genuine small gene. We present a multi-layer audit that tests this failure mode across genome annotation, transposon mutagenesis, CRISPR interference, homology, transcript mapping, proteomics, and population variation. We apply it to Rv2438A, a 92-codon conserved hypothetical open reading frame of Mycobacterium tuberculosis ranked first by our own dark-gene target screen. Rv2438A is superimposed on the essential NAD synthetase locus nadE: 44% lies within its coding sequence on the opposite strand, and the remainder covers its promoter and transcription start site. Consequently, three of five Himar1 sites lie within nadE, no CRISPRi guide can target Rv2438A without binding nadE, and the cross-species hit maps to the same nadE start junction. Rv2438A lacks its own transcription start site and is absent from every proteomic dataset that detects nadE. A genome-wide scan identifies six short, overlapping, uncharacterised loci among 3907 annotated genes, but only Rv2438A combines overlap and essentiality with non-detection across all proteomic datasets; rare genome-wide, it ranked first among screen hits. We provide an implementable audit workflow and a codon-position control, but measure the control's sensitivity as only two of five genes with attested protein, limiting it to confirmatory use. Overlap coordinates and neighbour-specific experimental resolution should therefore be reported before bacterial dark genes are prioritised.

CRISPR interference↗

PubMed1986

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.

Amino Acid Sequence↗

PubMed1988

Identification of the E5 open reading frame of human papillomavirus type 16.

Sequencing of the E5 open reading frame (ORF) of human papillomavirus type 16 revealed an additional nucleotide, a thymidine residue, at position 3903 compared with the original sequence (Seedorf et al., Virology 145:181-185, 1985). The additional T had two effects; first, in reading frame 2, in which the original E5 ORF was predicted, the additional T changed the reading frame downstream of position 3903 to create an ORF, which we designated E5, that terminated at position 4018 and potentially encoded a 52-amino-acid polypeptide. Secondly, in reading frame 3, a new ORF was created (positions 3807 to 4097), which we propose is the authentic papillomavirus type 16 E5 ORF. It contained a methionine residue and encoded an additional 82 amino acids. Both ORFs have been cloned into bacterial expression vectors (pATH), and the fusion proteins have been used to generate polyclonal antibodies in rabbits.

Amino Acid Sequence↗

PubMed1992

Indian hepatitis E virus shows a major deletion in the small open reading frame.

Enterically transmitted non-A, non-B hepatitis virus (HEV), the causative agent for sporadic and large epidemic outbreaks in developing countries, contains a positive-sense single-stranded RNA genome. The genome of the virus encodes three open reading frames (ORF1, ORF2, and ORF3). The gene segment corresponding to the small open reading frame (ORF3), overlapping between ORF1 and ORF2, was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) from a number of previously identified HEV-positive clinical specimens. A DNA fragment of 166 bp was consistently obtained from all the clinical specimens. This small fragment was cloned, sequenced, and found to contain an open reading frame encoding only 41 amino acid residues. Comparison of our results with that of geographically related Burma HEV suggests a major inframe deletion of 246 bp in the ORF3 of Indian strain. The protein encoded by ORF3 does not appear to be useful for early serodiagnosis as a synthetic peptide deduced from the truncated ORF3 failed to show any demonstrable immunoreactivity against HEV-infected acute phase sera in an enzyme-linked immunosorbent assay.

Antibody Formation↗

PubMed2006

Prokaryotes that grow optimally in acid have purine-poor codons in long open reading frames.

In nucleic acids the N-glycosyl bonds between purines and their ribose sugar moities are broken under acid conditions. If one strand of a duplex DNA segment were more vulnerable to mutation than the other, then the archaeon Picrophilus torridus, with an optimum growth pH near zero, could have adapted by decreasing the purine content of that strand. Yet, P. torridus has an optimum growth temperature near 60 degrees C, and thermophiles prefer purine-rich codons. We found that, as in other thermophiles, high growth temperature correlates with the use of purine-rich codons. The extra purines are often in third, non-amino acid determining, codon positions. However, as in other acidophiles, as open reading frame lengths increase, there is increased use of purine-poor codons, particularly those without purines in second, amino acid-determining, codon positions. Thus, P. torridus can be seen as adapting (a) to temperature by increasing its purines in all open reading frames without greatly impacting protein amino acid compositions, and (b) to pH by decreasing purines in longer open reading frames, thereby potentially impacting protein amino acid compositions. It is proposed that longer open reading frames, being larger mutational targets, have become less vulnerable to depurination by virtue of pyrimidine for purine substitutions.

Adaptation, Physiological↗

PubMed1985

Polymorphism of the 3' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Sequence of a 4.8 kb fragment of Saccharomyces cerevisiae chromosome II including three essential open reading frames.

The nucleotide sequence of a fragment of 4867 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three complete open reading frames. In addition to the already known gene RPB5, coding for a subunit shared by all three DNA directed RNA polymerases, two new open reading frames could be identified. YBR12.03 codes for a protein of 183 amino acids with homology to one of the proteins of the Bacillus subtilis riboflavin biosynthesis operon (RibG). Deletion mutants of YBR12.03 can germinate but stop growing after five to seven cell divisions on YPD. Supplementation with high concentrations of riboflavin does promote growth. YBR12.05 codes for a protein of 386 amino acids with homology to STI1, a stress-inducible protein of S. cerevisiae. Deletion mutants of YBR12.05 are not viable.

Amino Acid Sequence↗

PubMed1984

Predicted secondary structure similarity in the absence of primary amino acid sequence homology: hepatitis B virus open reading frames.

Proteins that are related evolutionarily may have diverged at the level of primary amino acid sequence while maintaining similar secondary structures. Computer analysis has been used to compare the open reading frames of the hepatitis B virus to those of the woodchuck hepatitis virus at the level of amino acid sequence, and to predict the relative hydrophilic character and the secondary structure of putative polypeptides. Similarity is seen at the levels of relative hydrophilicity and secondary structure, in the absence of sequence homology. These data reinforce the proposal that these open reading frames encode viral proteins. Computer analysis of this type can be more generally used to establish structural similarities between proteins that do not share obvious sequence homology as well as to assess whether an open reading frame is fortuitous or codes for a protein.

Amino Acid Sequence↗

PubMed1990

Developmental rearrangement of cyanobacterial nif genes: nucleotide sequence, open reading frames, and cytochrome P-450 homology of the Anabaena sp. strain PCC 7120 nifD element.

An 11-kbp DNA element of unknown function interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. In developing heterocysts the nifD element excises from the chromosome via site-specific recombination between short repeat sequences that flank the element. The nucleotide sequence of the nifH-proximal half of the element was determined to elucidate the genetic potential of the element. Four open reading frames with the same relative orientation as the nifD element-encoded xisA gene were identified in the sequenced region. Each of the open reading frames was preceded by a reasonable ribosome-binding site and had biased codon utilization preferences consistent with low levels of expression. Open reading frame 3 was highly homologous with three cytochrome P-450 omega-hydroxylase proteins and showed regional homology to functionally significant domains common to the cytochrome P-450 superfamily. The sequence encoding open reading frame 2 was the most highly conserved portion of the sequenced region based on heterologous hybridization experiments with three genera of heterocystous cyanobacteria.

Amino Acid Sequence↗

PubMed1995

Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

Amino Acid Sequence↗

PubMed2019

Distinct types of short open reading frames are translated in plant cells.

Genomes contain millions of short (<100 codons) open reading frames (sORFs), which are usually dismissed during gene annotation. Nevertheless, peptides encoded by such sORFs can play important biological roles, and their impact on cellular processes has long been underestimated. Here, we analyzed approximately 70,000 transcribed sORFs in the model plant Physcomitrella patens (moss). Several distinct classes of sORFs that differ in terms of their position on transcripts and the level of evolutionary conservation are present in the moss genome. Over 5000 sORFs were conserved in at least one of 10 plant species examined. Mass spectrometry analysis of proteomic and peptidomic data sets suggested that tens of sORFs located on distinct parts of mRNAs and long noncoding RNAs (lncRNAs) are translated, including conserved sORFs. Translational analysis of the sORFs and main ORFs at a single locus suggested the existence of genes that code for multiple proteins and peptides with tissue-specific expression. Functional analysis of four lncRNA-encoded peptides showed that sORFs-encoded peptides are involved in regulation of growth and differentiation in moss. Knocking out lncRNA-encoded peptides resulted in a decrease of moss growth. In contrast, the overexpression of these peptides resulted in a diverse range of phenotypic effects. Our results thus open new avenues for discovering novel, biologically active peptides in the plant kingdom.

Bryopsida↗

PubMed1993

The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein.

We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide. The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria. A mitochondrial polypeptide with an apparent molecular weight of 21,000 was detected with a polyclonal antibody raised against an ORF221 fusion protein. In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria. The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species.

Cell Fractionation↗

PubMed1997

Putative regulatory sequence in human papillomavirus type 16 E2 open reading frame.

A 114 bp fragment of the human papillomavirus type 16 (HPV16) E2 open reading frame (nt. 3142-3255) containing a putative estrogen responsive element (ERE) was amplified and cloned into pBLCAT2 plasmid in both sense (p159-4) and anti-sense (p164) orientation. The plasmids were transfected into human breast-cancer cell line MCF-7 containing estrogen receptor and the cultures were kept in the presence or absence of beta-estradiol. The chloramphenicol acetyltransferase (CAT) activity was not influenced by estrogen. However, a silencer effect was observed both in cultures transfected with p159-4 and p164 plasmids. We prepared and cloned synthetic fragments containing the putative ERE and failed to prove that the palindrome in the putative ERE was responsible for the silencer activity.

Base Sequence↗

PubMed1996

Genetic variation in open reading frame 2 of field isolates and laboratory strains of equine arteritis virus.

The open reading frame 2 (ORF2) of three laboratory strains, the live attenuated vaccine virus, and 18 field isolates of equine arteritis virus (EAV) from Europe and North America was sequenced. The ORF2 of EAV encodes the Gs protein that is abundantly expressed in infected cells but constitutes less than 2% of the virion protein mass. Variation of ORF2 among the isolates facilitated phylogenetic analysis that largely confirmed results of an earlier study based on sequence divergence of ORF5 of the same isolates of EAV, despite exposure of the proteins encoded by ORF2 (Gs) and ORF5 (GL) to potentially different selective pressures in vivo. The data indicate that the Gs protein is highly conserved between isolates, considerably more so than the GL protein, consistent with an important role of the Gs protein in virus replication.

Amino Acid Sequence↗